Pathogenic scarring and fibrosis represent important clinical problems with potentially serious consequences for patients, including impairment of normal tissue regeneration and neighboring tissue function. Our long-term goal is to identify the critical regulatory mechanisms that govern fibroblast differentiation Into myofibroblasts thereby developing novel strategies to control scarring and fibrosis. TGF- ~ is a central component of this mechanism and we recently determined that a focal adhesion protein, termed Hic-5, is induced by TGF-~ in normal human dermal fibroblasts;Hic-5 is also persistently expressed in hypertrophic scar myofibroblasts (HTSF). Importantly, Hic-5 is required for TGF-~ production in HTSF thereby regulating its own expression via the TGF-~ autocrine loop. Moreover, when Hic-5 is knocked down with RNAi the HTSF phenotype reverts to that of a normal dermal fibroblast supporting a therapeutic strategy of blocking fibrosis by targeting Hic-5 expression in myofibroblasts. We propose a two-year, ARRA sponsored, research program in which we test the hypothesis that TGF-~ induces Hic-5 expression through Rho GTPase-dependent pathway(s) thereby establishing an autocrine loop and a persistent myofibroblast phenotype. In Aim 1, we will define the intracellular pathways that regulate TGF-~-dependent, Hic-5 expression in myofibroblasts. The mechanisms through which Hic-5 is induced in normal human fibroblasts and perpetuated in pathogenic fibroblasts (HTSFs) will be determined. Experiments will be performed using pharmacological inhibitors, genetic silencing (RNAi) and cDNA reconstitution, in vitro. The levels of Hic-5 and candidate signaling molecules will be determined and changes in activity measured using phosphorylation-specific antibodies. In Aim 2, we will identify and analyze Hic-,5 regulated genes in hypertrophic scars. Expression (cDNA) arrays will be used to identify the genes products in HTSF that are regulated by Hic-5. These will be cross-referenced to genes that others have reported are markedly regulated in hypertrophic scars. Hic-5-dependent gene products identified from these cDNA arrays will also be ,analyzed as candidate genes that eitlier reguiate the TGF-~ autocrine loop or mediate specific myofibroblast functions, Key gene products will then be incorporated into quantitative PCR (qPCR) arrays and probed with RNA obtained from freshly resected hypertrophic scars. Immunohistology will be employed to confirm the expression and localization of these Hic-5- dependent gene products in scars. Findings obtained from this work will fill important gaps in understanding pathogenic myofibroblast regulation and suggest new therapeutic strategies to modulate fibrosis following serious burn or traumatic injury.